Nous apprécions grandement les contributions éditoriales par Cheryl Schaeberle. Cette recherche a été soutenue par des subventions Avon # 02-2009-093 et ​​02-2011-095 et NIEHS / NIH ES 08314 à AMS. Le contenu est de la seule responsabilité des auteurs et ne reflètent pas nécessairement les positions officielles de l&#39;Institut national des sciences de la santé de l&#39;environnement ou de la National Institutes of Health.

1. Préparation des réactifs <ol><li> Dissoudre le promégestone synthétique progestatif (R5020) et 17-β-estradiol (E2) dans l&#39;éthanol pour faire 10 -3 M solutions mères. Dissoudre la prolactine dans l&#39;eau déminéralisée distillée pour faire un / ml solution mère à 1 mg. Dissoudre l&#39;anti-oestrogène ICI 182,780 dans le DMSO pour faire un M solution mère à 10 -2. Stocker ces solutions à -20 ° C pendant jusqu&#39;à 6 mois. </li><li> dextran de charbon (CD) dépouillé sérum et moyennes CDFBS: <ol><li> Inactiver à la chaleur de sérum bovin fœtal (FBS) dans un bain d&#39;eau à 57 ° C pendant 30 min. Remarque: Utilisez uniquement la verrerie lavée à l&#39;acide pour le reste de ce protocole. </li><li> Calculer la quantité de charbon de bois nécessaire. Utilisation de 10% plus de volume que la quantité de sérum pour compenser le déplacement provoqué par le charbon, peser 5% en poids / volume de charbon actif en poudre. </li><li> Laver le charbon deux fois avec de l&#39;eau désionisée froide distillée en utilisant le même volume que la quantité de sérum. Pour chaque lavage centrifuge à 50 x g pendant 2 min pour sédimenter le charbon de bois. Retirer l&#39;eau entre les lavages. Ajouter 0,5% en poids / volume Dextran T-70 à la dernière charbon granulés. Re-suspendre avec distillée, eau déminéralisée et centrifuger à 100 g pendant 5 min. Aspirer l&#39;eau. </li><li> Ajouter le volume approprié de sérum au culot et remettre en suspension le culot de charbon dextran dans le sérum. Incuber, tout en roulant (6 RPM), à 37 ° C pendant 60 min. Centrifuger à 2000 g pendant 20 min. Si surnageant apparaît trouble, répéter centrifugation; Cependant, le sérum peut ne pas être tout à fait clair à ce moment, mais il sera effacé lors de la filtration. </li><li> Filtrer à travers un filtre de 0,80 microns, puis à nouveau à travers un filtre de 0,45 microns pour éliminer plus grand de particules. Enfin, le filtre-stériliser avec un filtre de 0,20 micron. Magasin CDFBS dans des tubes de verre ou T-25 flacons de culture filtré à -20 ºC. </li><li> Charcoal Dextran Stripped fœtal bovin contenant des médias (médias CDFBS): mélanger de 375 ml de phénol DMEM exempt de rouge et 125 ml de DMEM/ F-12. Retirer 37,5 ml et le remplacer par le même volume de sérum de veau fœtal charbon Dextran Stripped. Ajouter 10 6 U / ml de pénicilline et de la L-glutamine à une concentration finale de 2 mM. Le support obtenu est 75% de DMEM, 25% de DMEM / F-12, de 7,5% à charbon dextran dépouillée de FBS, 2 mM de L-glutamine, et 10 6 U / ml de pénicilline. </li></ol></li><li> Préparez 10X PBS, NaOH 1N et de l&#39;eau déminéralisée distillée. Préparer un volume suffisant de chaque selon le volume de la solution de collagène est nécessaire. Stériliser toutes les solutions de filtrage. Stocker le 10x PBS et de l&#39;eau déminéralisée distillée stérile à 4 ° C pendant jusqu&#39;à 1 mois. Ne pas conserver la solution de NaOH 1N. </li><li> Préparer la solution de collagène à 1 mg / ml de concentration finale selon le protocole de &quot;procédure de gélification suppléant de collagène I, queue de rat&quot; fourni par le fabricant. Chaque gel est constitué de 1,5 ml d&#39;une solution de collagène. Par exemple, préparer 18,5 ml (0,5 ml pour tenir compte des pertes au cours de pipetage) d&#39;une solution de collagène pour préparer12 gels. Utiliser la solution de collagène immédiatement ou tenir sur de la glace jusqu&#39;à 2-3 heures. </li><li> Carmine Alum colorant: <ol><li> Mélanger 1 g Carmine et 2,5 g de sulfate d&#39;aluminium de potassium dans 500 ml d&#39;eau distillée. Faire bouillir tout en agitant pendant 20 min. Regardez attentivement pour éviter de déborder. Ajuster le volume final de retour à 500 ml d&#39;eau distillée supplémentaire. </li><li> Ajouter un cristal de thymol de l&#39;activité antimicrobienne et couvrir la bouteille avec du papier d&#39;aluminium pour le protéger de la lumière. Filtrer à travers un filtre en papier de qualité laboratoire avant chaque utilisation. Réutiliser la solution de carmin pour un maximum de trois mois. Magasin à la température ambiante, à l&#39;abri de la lumière. </li></ol></li><li> solution de collagénase: dissoudre la collagénase dans du milieu DMEM / F12 à une concentration de 0,125% p / v. Ne rangez pas cette solution. </li></ol> 2. 3D culture de cellules T47D dans Queue Rat collagène de type 1 et Gels Traitement hormonal Remarque: garder pipettes sérologiques stériles et des embouts de pipette à 4 ° C. <ol><li> Prepare une suspension à cellule unique et d&#39;effectuer une numération cellulaire. des cultures de cellules de Split T47D avant qu&#39;ils atteignent 70% de confluence. Centrifuger le volume qui contient la quantité de cellules nécessaires. Considérer que l&#39;un de gel devrait contenir environ 75.000 T47D cellules. </li><li> En utilisant une pipette à froid, remettre en suspension le culot cellulaire dans le volume approprié de collagène. Considérons que chaque gel est constitué de 1,5 ml de collagène. Mélanger les cellules et le collagène avec précaution pour éviter l&#39;introduction de bulles. </li><li> Pour éviter l&#39;effet de l&#39;électricité statique sur la distribution de cellules, brosser la frontières, haut et en bas de la plaque de 12 puits avec une brosse antistatique réducteur après déballer la plaque. Pour éviter l&#39;accumulation de charge statique lors du contact avec la surface de travail de la hotte, placer cette plaque sur le dessus d&#39;une autre plaque de 12 puits (plaque vierge) qui a également été brossé avec le brush.Gently statique de réduction, versez 1,5 ml du mélange dans chaque puits de la plaque de 12 puits. </li><li> Placer la plaque dans une étuve à 37 ° C pendant 30 min. Retirer ee plaque de l&#39;incubateur et le replacer dans la hotte. </li><li> En utilisant une pointe stérile p200, détacher soigneusement le gel dans un mouvement circulaire de la frontière du puits. Le gel doit également se détacher du fond du puits. </li><li> Diluer hormones dans les médias CDFBS à être ajoutés aux puits. R5020 et utiliser E2 à une concentration finale de 10 -10 M et de la prolactine à une 10 -7 M à une concentration finale. Comme contrôle, utiliser les médias CDFBS sans hormones ajoutées, mais qui contient encore le plus grand volume de DMSO égale aux stocks d&#39;hormones utilisées. Veiller à ce que chaque puits contient 1,5 ml de milieu. Placer les cultures 3D à l&#39;intérieur de 37 ° C, 5% CO 2, 100% Taux d&#39;humidité de la culture tissulaire de l&#39;incubateur. Maintenir les cultures pour 1 ou 2 semaines, en changeant les médias tous les 2 jours. </li></ol> 3. Traitement Gel pour toute Mounts <ol><li> Retirer les milieux de culture et d&#39;ajouter 1,5 ml de PBS à chaque puits. </li><li> Transférer le gel à une diapositive. Couper le gel en deux à l&#39;aide d&#39;une courbelame chirurgicale d. Continuer le traitement avec une moitié et sauver l&#39;autre moitié pour l&#39;analyse histologique. </li><li> Transférer le gel de la moitié de toute la montagne à une diapositive. Laissez gel sec pendant 5 min, puis transférer la diapositive à un bocal de Coplin contenant 10% de formol. Remarque: toutes les incubations doivent être effectués à la température ambiante. </li><li> Fixer des gels dans un bocal de Coplin sur un agitateur O / N. </li><li> Laver les lames deux fois avec PBS pendant 10 min chacun sur shaker. Transfert glisse à 70% EtOH; incuber pendant 1 heure sur un agitateur. diapositives de transfert aux Carmine Alum O / N. </li><li> Le lendemain, déshydrater diapositives sur un agitateur à 70% de EtOH, EtOH à 95% et 100% EtOH pendant 1 heure chacune d&#39;elles. Transfert glisse au xylène deux fois pendant 1 heure chacun et continuer à shaker. Utilisez milieu de montage à base de toluène de monter les gels et 1,5 mm Lamelles épaisses. </li><li> Examiner montures entières sous une lumière ordinaire (microscope ou stéréoscope). Pour une analyse plus détaillée de la forme et de la lumière en utilisant la microscopie confocale et de visualiser Carmine colorant avec HeNe 633 nm laser. </li></ol>4. Traitement Gel pour le traitement de l&#39;analyse histologique <ol><li> Transférer la moitié restante du gel dans un flacon en verre de 20 ml contenant 10% de formol et de fixer sur un agitateur O / N. </li><li> Lavez gels deux fois avec PBS pendant 10 min chacun sur shaker. La place des gels dans des cassettes de traitement pour inclusion dans la paraffine. </li><li> gels de transfert à 70% EtOH, incuber pendant 1 h et suivent avec 95% EtOH pendant 1 heure, changent à nouveau de 95% EtOH pendant 1 heure, 100% EtOH pendant 1 heure, et se transforment en frais EtOH à 100% pendant 1 heure. À ce stade, les gels peuvent être stockés dans EtOH à 100% O / N à la température ambiante ou incubées avec du xylène pendant 1 heure, puis à nouveau dans le xylène frais pendant 1 heure. </li><li> cassettes de transfert de paraffine pendant 1 heure à 60 ° C dans un incubateur à vide, répéter avec de la paraffine frais deux fois plus. </li><li> Remplir les moules en plastique avec de la paraffine propre. Ouvrez la cassette, enlever le gel et le placer dans le moule contenant la paraffine plastique. Remplir avec de la paraffine et ajoutez haut de cassette contenant l&#39;étiquette sur le dessus et laisser bloc cool. </li><li> Retirez le plastic moule et la section du bloc en utilisant un microtome. Obtenez 5 um sections d&#39;épaisseur pour coloration HE et immunocytochimie. </li></ol> 5. extraction de cellules à partir de cellules à partir de gels en utilisant un traitement collagénase <ol><li> Retirer le gel du puits et le transférer à une boîte de Pétri contenant du PBS. brièvement de gel rinçage. </li><li> Transférer le gel dans un tube de 15 ml contenant 2 ml de 0,125% de collagénase. Introduire à la pipette de haut en bas 4-5 fois pour briser le gel en petits morceaux. </li><li> Incuber à 37 ° C pendant 25 min (jusqu&#39;à ce que le gel se soit dissous et les cellules sont en suspension) </li><li> Ajouter 2 ml de sérum supplémenté le milieu de culture. Recueillir les cellules par centrifugation (1000 x g, 5 min), surnageant rejet, remettre en suspension les cellules et à compter. </li></ol>

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	<li>Barnes, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+single+cells+to+tissues:+interactions+between+the+matrix+and+human+breast+cells+in+real+time.">From single cells to tissues: interactions between the matrix and human breast cells in real time.</a> PLoS ONE. 9, e93325 (2014).</li><li>Berthois, Y., Katzenellenbogen, J. A., Katzenellenbogen, B. 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M., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+collagen+reorganization+on+mammary+epithelial+morphogenesis+in+a+3D+culture+model.">The role of collagen reorganization on mammary epithelial morphogenesis in a 3D culture model.</a> Biomaterials. 31, 3622-3630 (2010).</li><li>Dhimolea, E., Soto, A. M., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+epithelial+tissue+morphology+is+affected+in+3D+cultures+by+species-specific+collagen-based+extracellular+matrix.">Breast epithelial tissue morphology is affected in 3D cultures by species-specific collagen-based extracellular matrix.</a> J.Biomed.Mat.Res.A. 100, 2905-2912 (2012).</li><li>Krause, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+regulation+of+breast+tubulogenesis+using+extracellular+matrix+composition+and+stromal+cells.">Dual regulation of breast tubulogenesis using extracellular matrix composition and stromal cells.</a> Tissue Eng Part A. 18, 520-532 (2012).</li><li>Krause, S., Maffini, M. V., Soto, A. M., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+3D+in+vitro.+culture+model+to+study+stromal-epithelial+interactions+in+the+mammary+gland.">A novel 3D in vitro. culture model to study stromal-epithelial interactions in the mammary gland.</a> Tissue Eng.Part C Methods. 14, 261-271 (2008).</li><li>Krause, S., Maffini, M. V., Soto, A. M., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microenvironment+determines+the+breast+cancer+cells'+phenotype:+organization+of+MCF7+cells+in+3D+cultures.">The microenvironment determines the breast cancer cells' phenotype: organization of MCF7 cells in 3D cultures.</a> BMC Cancer. 10, 263-275 (2010).</li><li>Lee, G. Y., Kenny, P. A., Lee, E. H., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+models+of+normal+and+malignant+breast+epithelial+cells.">Three-dimensional culture models of normal and malignant breast epithelial cells.</a> Nat.Methods. 4, 359-365 (2007).</li><li>Martinson, H. A., Jindal, S., Durand-Rougely, C., Borges, V. F., Schedin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing-like+immune+program+facilitates+postpartum+mammary+gland+involution+and+tumor+progression.">Wound healing-like immune program facilitates postpartum mammary gland involution and tumor progression.</a> Int.J.Cancer. , (2014).</li><li>Soto, A. M., Murai, J. T., Siiteri, P. K., Sonnenschein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+proliferation:+evidence+for+negative+control+on+estrogen-sensitive+T47D+human+breast+cancer+cells.">Control of cell proliferation: evidence for negative control on estrogen-sensitive T47D human breast cancer cells.</a> Cancer Res. 46, 2271-2275 (1986).</li><li>Speroni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+regulation+of+epithelial+organization+in+a+3D+breast+tissue+culture+model.">Hormonal regulation of epithelial organization in a 3D breast tissue culture model.</a> Tissue Eng.Part C Methods. 20, 42-51 (2014).</li><li>Vignon, F., Bardon, S., Chalbos, D., Rochefort, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antiestrogenic+effect+of+R5020,+a+synthetic+progestin+in+human+breast+cancer+cells+in+culture.">Antiestrogenic effect of R5020, a synthetic progestin in human breast cancer cells in culture.</a> J.Clin.Endocrinol.Metab. 56, 1124-1130 (1983).</li></ol>

The authors have nothing to disclose.

Here, we describe a hormone-sensitive 3D culture model to test the action of hormones on breast epithelium. The response to hormones can be assessed at the tissue morphology, cell proliferation and gene expression levels 12. One limitation of this technique is that visualization during the culture period is restricted to light microscopy since the cultures are grown in a plastic bottom plate. The 3D culture system could be adapted to glass bottom plates to allow for live imaging of the cultures 1.</...

Contrairement cultures 2D standard, culture cellulaire 3D modèles de substitution permettent l&#39;étude du comportement des cellules épithéliales dans un contexte physiologiquement pertinente, l&#39;un ressemble à un tissu. cultures 3D de la glande mammaire ont permis d&#39;élucider de nombreux aspects du développement de la glande mammaire et la néoplasie. Cependant, la plupart des modèles de culture 3D actuellement ne conviennent pas pour étudier l&#39;action des hormones parce que les lignées de cellules épithéliales humaines utilisées pour la tâche manquent de l&#39;expression du récepteur d&#39;hormone 6,7,9. Nous décrivons ici un modèle de culture 3D de l&#39;épithélium du sein humain qui est approprié pour étudier l&#39;action de l&#39;hormone 12. Ce modèle utilise la ligne disponible dans le commerce hormono-sensible humain epithelial du sein cellulaire T47D 3,11,13, qui ont été à l&#39;origine dérivée d&#39;une effusion pleurale provenant d&#39;une patiente de 54 ans femelle avec un carcinome canalaire infiltrant du sein. Nous utilisons rat de type queue de collagène 1 comme matrice. Ce culte 3Dmodèle de ure est approprié pour l&#39;étude de l&#39;action des trois principales hormones mammotropic (estradiol, promégestone (un analogue de la progestérone), et de prolactine) sur les cellules épithéliales mammaires humaines. Hormone induit morphologie épithéliale peut être évaluée quantitativement au fil du temps par analyse morphométrique 12. Une densité de semis appropriée permet à ces cultures 3D pour être conservés pendant 2 semaines. A cette époque, le développement de structures est suffisant pour une évaluation quantitative robuste de l&#39;action des hormones sur la morphologie épithéliale. Les gels peuvent également être récoltées à des moments antérieurs pour la prolifération cellulaire et les analyses d&#39;expression génique. En outre, ce modèle est adapté à tester les effets d&#39;un traitement hormonal séquentiel; par exemple, après le traitement avec l&#39;oestradiol pendant la première semaine et son remplacement par un autre hormone / combinaison d&#39;hormones au cours de la semaine suivante. L&#39;effet des composés oestrogéniques et anti-œstrogènes, tels que ICI 182,780, peut également être Stumortes en utilisant ce modèle de culture de 12 3D.

<table><tbody><tr><td>12-well Tissue Culture Plates (Falcon)</td><td>Fisher Scientific</td><td>08-772-29</td><td></td></tr><tr><td>15 ml polystyrene conical Tubes</td><td>Fisher Scientific</td><td>14-959-49D</td><td></td></tr><tr><td>Activated Charcoal</td><td>Sigma</td><td>C-5510</td><td></td></tr><tr><td>Carmine Alum</td><td>Sigma</td><td>C1022-100G</td><td></td></tr><tr><td>Collagenase, Type 3</td><td>Worthington</td><td>S0C11784</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td>LSM510</td><td>Equiped with HeNe 633nm laser</td></tr><tr><td>Dextran T-70</td><td>Abersham/Pharmacia</td><td>17-0280-01</td><td></td></tr><tr><td>DMEM/F-12, HEPES, no phenol red&amp;nbsp;</td><td>Life Technologies</td><td>11339-021</td><td>Phenol red-free media for hormone use</td></tr><tr><td>DMEM, low glucose, pyruvate, no glutamine, no phenol red</td><td>Life Technologies</td><td>11054-020</td><td>Phenol red-free media for hormone use</td></tr><tr><td>17-&amp;beta;-Estradiol</td><td>EMD Millipore</td><td>3301</td><td>Dissolved in Ethanol</td></tr><tr><td>Ethanol</td><td>Koptec</td><td>V1001</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SH30070.03</td><td>For use with hormones, must be Charcoal Dextran stripped&amp;nbsp;</td></tr><tr><td>Filters (115 ml)</td><td>Nalgene</td><td>380-0080, 245-0045, 120-0020</td><td>0.88, 0.45, 0.20 micron, respectively&amp;nbsp;</td></tr><tr><td>Formalin, 10%</td><td>Fisher Scientific</td><td>SF93-20</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>ICI 182,780 (fulvestrant)</td><td>Sigma Aldrich</td><td>I4409-25MG</td><td>Dissolved in DMSO</td></tr><tr><td>Microtome&amp;nbsp;</td><td>Leica</td><td>RM2155</td><td></td></tr><tr><td>Tissue embedding media</td><td>McCormick Scientific</td><td>39502004</td><td></td></tr><tr><td>Penicillin</td><td>Sigma</td><td>7794-10MU</td><td>Dissolved in 10 ml of distilled deionized water</td></tr><tr><td>Permount</td><td>Fisher Scientific</td><td>SP15-500</td><td></td></tr><tr><td>Phosphate Buffered Saline pH 7.4</td><td>Sigma Aldrich</td><td>P3813-10PAK</td><td></td></tr><tr><td>Prolactin</td><td>Sigma Aldrich</td><td>L4021-50UG</td><td>Dissolved in distilled deionized water</td></tr><tr><td>Promegestone</td><td>Perkin Elmer</td><td>NLP004005MG</td><td>Dissolved in Ethanol</td></tr><tr><td>Rat-Tail Collagen&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>354236</td><td>Lots may contain varying concentrations, note accordingly</td></tr><tr><td>Scalpel</td><td>Miltex</td><td>4311</td><td></td></tr><tr><td>Semi-enclosed Benchtop Tissue Processor</td><td>Leica</td><td>TP1020</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma Aldrich</td><td>S5881</td><td>Prepare 1N NaOH stock</td></tr><tr><td>StaticMaster Anti-static brush&amp;nbsp;</td><td>Amstat</td><td>C3500</td><td></td></tr><tr><td>Stripette Serological Pipettes&amp;nbsp;</td><td>Corning&amp;nbsp;</td><td>4101</td><td></td></tr><tr><td>T-25 flasks</td><td>Corning&amp;nbsp;</td><td>430168</td><td></td></tr><tr><td>Tissue Cassettes</td><td>Fisher Scientific</td><td>15-200-403E</td><td></td></tr><tr><td>Wheaton Vials, Glass, 20 ml</td><td>Fisher Scientific</td><td>03-341-25D&amp;nbsp;</td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>VWR</td><td>95057-822</td><td></td></tr></tbody></table>

a hormone-responsive 3d culture model of the human mammary gland epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

La figure 1 résume la procédure pour la préparation des cultures 3D hormono-sensibles. structures epitheliales sont observés dans les montures de gels entiers cultivés pendant 2 semaines en présence de E2 seul ou en combinaison avec d&#39;autres hormones. Des cellules ou des groupes de 2-3 cellules simples sont présents seulement lorsque aucun hormones sont ajoutées au milieu de culture (milieu CDFBS) (figure 2). Cette condition sert de contrôle négatif. <p class="jove_con...

Watch this Scientific Journal Video about A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium at JoVE.com

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

Un modèle 3D Culture hormone-sensibles de la glande mammaire humaine épithélium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

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8.7K vues.  Tufts University School of Medicine. We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

Vidéo: A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation.
The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins &#946;-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed &#34;domes&#34;.
The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce &#946;-casein and WAP.
Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

We describe techniques for differentiation induction of two breast epithelial lines, HC11 and EpH4. While both require fetal calf serum, insulin, and prolactin to produce milk proteins, EpH4 cells can fully differentiate into mammospheres in three-dimensional culture. These complementary models are useful for signal transduction studies of differentiation and neoplasia.

Différenciation des cellules Epithéliales De l’épithélial de souris HC11 et EpH4

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction ...

Recherche

Recherche en cancérologie

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

A Tissue Culture Modèle tridimensionnel pour étudier primaire moelle osseuse humaine et de ses affections malignes

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Médecine

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been provided by studies using these models. Cell lines have provided an understanding of the deregulation of molecular signaling associated with breast tumorigenesis, while rodent models are widely used to study cellular and molecular characteristics of breast cancer in vivo. The establishment of 3D cultures of breast epithelial and cancerous cells aids in bridging the gap between in vivo and in vitro models by mimicking the in vivo conditions in vitro. This model can be used to understand the deregulation of complex molecular signaling events and the cellular characteristics during breast carcinogenesis. Here, a 3D culture system is modified to study a phospholipid mediator-induced (Platelet Activating Factor, PAF) transformation. Immunomodulators and other secreted molecules play a major role in tumor initiation and progression in the breast. In the present study, 3D acinar cultures of breast epithelial cells are exposed to PAF exhibited transformation characteristics such as loss of polarity and altered cellular characteristics. This 3D culture system will assist in shedding light on genetic and/or epigenetic perturbations induced by various small molecule entities in the tumor microenvironment. Additionally, this system will also provide a platform for the identification of novel as well as known genes that may be involved in the process of transformation.

The present protocol describes the setting up of 3D 'on top' cultures of a non-transformed breast epithelial cell line, MCF10A, that has been modified to study Platelet Activating Factor (PAF) induced transformation. Immuno-fluorescence has been used to assess the transformation and is discussed in detail.

Transformation induite par un médiateur de phospholipides dans des cultures tridimensionnelles

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Trois Cultures dimensionnelles: un outil pour étudier l&#39;architecture normale Acinaire vs transformation maligne des cellules mammaires

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Quantification du cancer du sein cellulaire envahissement aide d&#39;un (3D) du modèle en trois dimensions

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

Génération et imagerie d’organoïdes épithéliaux de souris et humains à partir de tissus mammaires normaux et tumoraux sans passage

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Ingénierie en trois dimensions des tissus épithéliaux incorporés au sein de la matrice extracellulaire

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Bio-ingénierie

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Génération d’organoïdes mammaires mosaïques par trypsinisation différentielle

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Biologie du développement

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

Une<em&gt; Ex vivo</em&gt; Modèle pour étudier action hormone dans le sein humain

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Isolement des cellules épithéliales mammaires à partir de trois dimensions de cellules mixtes Sphéroïde Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Un modèle 3D Culture hormone-sensibles de la glande mammaire humaine épithélium

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